The goal of this project is to obtain a basic science information regarding therapeutic radiopharmaceuticals that are based on targeting the decay of Auger-electron-emitting radionuclides to specific sequences in genetic DNA. The principal innovation in our approach is that it is the specific DNA sequence of a gene within the genome of a cell that becomes the target of this "radiotherapy", not the total DNA of that cell. Gene-targetted "radiotherapy" optimally utilizes the sub-nanometer effect range of the Auger-electron-emitting radionuclides to allow targeting of most of the radiodamage to a selected gene sequence while producing minimal damage to the rest of the genome and to other cell components. &#8232;This approach requires a carrier molecule that exhibits enough specificity for a selected DNA sequence to deliver the radionuclide to that specific sequence and not to other sites in the genome. As our initial carrier molecule, we selected short synthetic oligonucleotides that are able to form a sequence-specific triple helix with the target DNA sequence, so-called triplex-forming oligonucleotides (TFO). We demonstrated the ability of 125I-TFO-NLS conjugates to produce double strand breaks in a specific site in the human multidrug resistance (mdr1) gene within cells. Currently we are developing a new generation of DNA sequence- and DNA structure-specific molecules, so-called peptide nucleic acids (PNA) consisting of DNA bases connected by peptide backbone. We are working to optimize the delivery of short DNA and PNA molecules into the cell nucleus using a gamma-H2AX foci-formation assay detecting the damage to target DNA. [unreadable] We studied the distribution of DNA strand breaks produced by decay of 125I, and the repair of these breaks by protein extracts from mammalian cells. We found that the repair of the radiodecay-produced breaks was orders of magnitude less effective than that of the breaks produced by restriction enzymes; and it was always associated with deletions at the target site. We have begun to map and to define the complete spectrum and distribution of DNA lesions associated with Auger-electron-emitter-induced DSBs. Initial results indicate that these DSBs are associated with base damage and other types of DNA lesions proximal and distal to the DSB. Using our in vitro DSB repair assay, we have shown such damaged DNA structures to be strong inhibitors of human NHEJ repair. [unreadable] Using DNA microarray methodology, we showed that in normal human fibroblasts nuclear DNA-targeted decays of 125I produce about ten times fewer differentially expressed genes than whole cell exposures from gamma irradiation at comparable doses. These results suggest that the effects of ionizing radiation on changes in global gene expression depend upon the distribution and rate of energy deposition in the cell. We are completing studies using gene-expression analyses to examine the cellular responses to the DNA damage produced by Auger-decay effects in comparison with the gene expression patterns following external gamma irradiations of human embryonic stem cells.